PROJECT SUMMARY/ABSTRACT Leukemia therapy remains challenging and options are limited for a large fraction of patients who relapse. Growing evidence suggests that the therapy outcome and failure in myeloid leukemias are intimately linked to properties of leukemic stem cells (LSCs). Quiescence is a fundamental property shared between LSCs and normal hematopoietic stem cells (HSCs). Thus, targeting quiescent leukemic stem cells is essential for a successful long-lasting leukemia therapy. Achieving this goal requires identification of building blocks of stem cell quiescence and an in-depth understanding of mechanisms that wire them together. By exploiting mitochondrial heterogeneity we identified discrete deeply quiescent and potent subsets of mouse and human HSCs. This led us to our discovery that lysosomal activity is heterogeneous in HSCs and key in maintaining their quiescence. We find lysosomes retain damaged mitochondria and that they are critical to the maintenance of HSC quiescence and metabolism. We have extended these studies to myeloid leukemias, using both a mouse model of pre-leukemia and leukemia as well as leukemic patients’ samples and find that leukemic stem cell populations are heterogeneous distinctively in their lysosomal and mitochondrial properties. Based on our combined results we propose to investigate the implications of lysosomal heterogeneity for LSC isolation, generation and maintenance. In Aim 1, we will take advantage of combined lysosomal and mitochondrial heterogeneity to select LSCs subsets for further investigating their distinct function related to their lysosomal and mitochondrial alterations; in Aim 2, we will investigate the modulation of lysosomal-mediated metabolic pathways in LSC subsets; and in Aim 3, we will investigate the potential of dysregulated lysosomes in mediating the generation of pre-leukemic stem cells. Altogether these studies are likely to improve our approaches for isolating LSCs and our understanding of LSC generation and maintenance.